Neisseria gonorrhoeae, is a gram-negative diplococcal bacterial pathogen which is the causative organism of the sexually transmitted disease (STD) known as gonorrhoea. Although gonorrhoea is an ancient disease first described by Galen in AD 150, it is still a major STD of humans. Failure to detect and treat Neisseria gonorrhoeae infection can allow the disease to progress to a serious systemic infection that affects the heart, joints, meninges, eyes and pharynx. Thus, early definitive diagnosis can assist treatment of the disease and prevent the serious complications that can arise as a result of this bacterial infection.
Although polymerase chain reaction (PCR) is the method of choice for routine detection of Neisseria gonorrhoeae, PCR has limitations and bacterial isolation has remained the gold standard for definitive diagnosis. This is largely because N. gonorrhoeae shares much sequence homology with other Neisseria species, including N. meningitidis, and in addition, contains many non-conserved sequences (Palmer et al., 2003, J. Clin. Microbiol. 41 835-7). Thus, there is a potential for both false-positive and false-negative results to occur when using PCR for routine detection of N. gonorrhoeae. In both situations the consequences may be significant. From a public health perspective, false-negative results may allow unchecked spread of the disease whereas false-positive results can have considerable social ramifications for patients.
The Roche Cobas Amplicor system (Roche Diagnostics, Australia) is a PCR assay widely used for the detection of N. gonorrhoeae. Its appeal lies in its ability to simultaneously detect N. gonorrhoeae, Chlamydia trachomatis and the presence of inhibiting substances, while also carrying United States Food and Drug Administration (FDA) approval. However, the Cobas Amplicor system does have limitations. Most, notably, its N. gonorrhoeae assay is known to cross react with some strains of commensal Neisseria species, including N. subflava, N. cinerea, N. flavescens, N. lactamica and N. sicca (Palmer et al., 2003, supra; Farrell, 1999, J. Clin. Microbiol. 37 386-90). Consequently, there is a need to use a second PCR assay to confirm Cobas Amplicor positive results. In response, clinical laboratories have adopted in-house confirmatory assays.
To date, the most common target for in-house confirmatory tests has been the cryptic plasmid (cppB) gene of N. gonorrhoeae, with several such protocols having been described (Ho et al., 1992, J. Clin Pathol. 45 439-442; Farrell, 19995 supra; Whiley et al., 2002, Diagn. Microbiol. Infect. Dis. 42 85-9; Tabrizi et al., 2004, Sex. Trans. Infect 80 68-71). In particular, a LightCycler based cppB PCR (cppB-LC) assay has been developed for conformation of Cobas N. gonorrhoeae positive specimens (Whiley et al., 2002, supra). However, serious concerns have now been raised over the sensitivity and specificity of N. gonorrhoeae assays targeting the cppB gene. Studies conducted in both the United Kingdom and Australia have identified N. gonorrhoeae isolates lacking the cppB gene (Palmer et al. 2003, supra, Ottawa; A cluster of culture-positive, but PCR false negative infections with Neisseria gonorrhoeae. Tapsall et al., Abstract 0129. 15th Biennial Congress of the International Society for Sexually Transmitted Diseases Research ISSTDR). Therefore, laboratories targeting this gene run the risk of false-negative results. In addition, the cppB gene could be present in commensal Neisseria strains, including N. cinerea, and so could also produce false-positive results (Palmer et al. 2003, supra).
Cross-reaction is a significant problem for gonococcal nucleic acid-based diagnostic testing and horizontal genetic exchange in the Neisseria genus is the major source of these cross-reactions (Johnson et al., 2002, MMWR Recomm. Rep 18 1-38). Furthermore, gonococcal tests are used on non-sterile sites and other Neisseria strains may frequently be found in such sites.
PCR detection of the porA gene has been used to detect. Neisseria meningitidis (Glustein et al., 1999, Molecular Diagnosis 4 233-9), partly due to an assumption that is gene is absent in commensal Neisseria (Feavers & Maiden, 1998, Mol. Microbiol. 30 647-656). Furthermore, in such assays, cross-reaction (or the potential for cross-reaction) is not a significant threat as these tests are used on sterile sites, including blood and CSF.
The only other Neisseria species where a porA sequence has been identified is N. gonorrhoeae, which has a porA pseudogene of considerable sequence similarity to the N. meningitidis porA gene (Feavers & Maiden, 1998, supra). However, it is not clear whether this pseudogene is present in all strains of N. gonorrhoeae, nor has its absence in commensal strains been verified.